NADPH Oxidases and Oxidative Stress in the Pathogenesis of Atrial Fibrillation.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and its prevalence increases with age. The irregular and rapid contraction of the atria can lead to ineffective blood pumping, local blood stasis, blood clots, ischemic stroke, and heart failure. NADPH oxidases (NOX) and mitochondria are the main sources of reactive oxygen species in the heart, and dysregulated activation of NOX and mitochondrial dysfunction are associated with AF pathogenesis. NOX- and mitochondria-derived oxidative stress contribute to the onset of paroxysmal AF by inducing electrophysiological changes in atrial myocytes and structural remodeling in the atria. Because high atrial activity causes cardiac myocytes to expend extremely high energy to maintain excitation-contraction coupling during persistent AF, mitochondria, the primary energy source, undergo metabolic stress, affecting their morphology, Ca2+ handling, and ATP generation. In this review, we discuss the role of oxidative stress in activating AF-triggered activities, regulating intracellular Ca2+ handling, and functional and anatomical reentry mechanisms, all of which are associated with AF initiation, perpetuation, and progression. Changes in the extracellular matrix, inflammation, ion channel expression and function, myofibril structure, and mitochondrial function occur during the early transitional stages of AF, opening a window of opportunity to target NOX and mitochondria-derived oxidative stress using isoform-specific NOX inhibitors and mitochondrial ROS scavengers, as well as drugs that improve mitochondrial dynamics and metabolism to treat persistent AF and its transition to permanent AF.

Neonatal Scn1b-null mice have sinoatrial node dysfunction, altered atrial structure, and atrial fibrillation.

Loss-of-function (LOF) variants in SCN1B, encoding the voltage-gated sodium channel ß1/ß1B subunits, are linked to neurological and cardiovascular diseases. Scn1b-null mice have spontaneous seizures and ventricular arrhythmias and die by approximately 21 days after birth. ß1/ß1B Subunits play critical roles in regulating the excitability of ventricular cardiomyocytes and maintaining ventricular rhythmicity. However, whether they also regulate atrial excitability is unknown. We used neonatal Scn1b-null mice to model the effects of SCN1B LOF on atrial physiology in pediatric patients. Scn1b deletion resulted in altered expression of genes associated with atrial dysfunction. Scn1b-null hearts had a significant accumulation of atrial collagen, increased susceptibility to pacing induced atrial fibrillation (AF), sinoatrial node (SAN) dysfunction, and increased numbers of cholinergic neurons in ganglia that innervate the SAN. Atropine reduced the incidence of AF in null animals. Action potential duration was prolonged in null atrial myocytes, with increased late sodium current density and reduced L-type calcium current density. Scn1b LOF results in altered atrial structure and AF, demonstrating the critical role played by Scn1b in atrial physiology during early postnatal mouse development. Our results suggest that SCN1B LOF variants may significantly impact the developing pediatric heart.

Tetrodotoxin-Sensitive Neuronal-Type Na+ Channels: A Novel and Druggable Target for Prevention of Atrial Fibrillation.

Background Atrial fibrillation (AF) is a comorbidity associated with heart failure and catecholaminergic polymorphic ventricular tachycardia. Despite the Ca2+-dependent nature of both of these pathologies, AF often responds to Na+ channel blockers. We investigated how targeting interdependent Na+/Ca2+ dysregulation might prevent focal activity and control AF. Methods and Results We studied AF in 2 models of Ca2+-dependent disorders, a murine model of catecholaminergic polymorphic ventricular tachycardia and a canine model of chronic tachypacing-induced heart failure. Imaging studies revealed close association of neuronal-type Na+ channels (nNav) with ryanodine receptors and Na+/Ca2+ exchanger. Catecholamine stimulation induced cellular and in vivo atrial arrhythmias in wild-type mice only during pharmacological augmentation of nNav activity. In contrast, catecholamine stimulation alone was sufficient to elicit atrial arrhythmias in catecholaminergic polymorphic ventricular tachycardia mice and failing canine atria. Importantly, these were abolished by acute nNav inhibition (tetrodotoxin or riluzole) implicating Na+/Ca2+ dysregulation in AF. These findings were then tested in 2 nonrandomized retrospective cohorts: an amyotrophic lateral sclerosis clinic and an academic medical center. Riluzole-treated patients adjusted for baseline characteristics evidenced significantly lower incidence of arrhythmias including new-onset AF, supporting the preclinical results. Conclusions These data suggest that nNaVs mediate Na+-Ca2+ crosstalk within nanodomains containing Ca2+ release machinery and, thereby, contribute to AF triggers. Disruption of this mechanism by nNav inhibition can effectively prevent AF arising from diverse causes.

Mitochondrial and Sarcoplasmic Reticulum Interconnection in Cardiac Arrhythmia.

Ca2+ plays a pivotal role in mitochondrial energy production, contraction, and apoptosis. Mitochondrial Ca2+-targeted fluorescent probes have demonstrated that mitochondria Ca2+ transients are synchronized with Ca2+ fluxes occurring in the sarcoplasmic reticulum (SR). The presence of specialized proteins tethering SR to mitochondria ensures the local Ca2+ flux between these organelles. Furthermore, communication between SR and mitochondria impacts their functionality in a bidirectional manner. Mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniplex is essential for ATP production and controlled reactive oxygen species levels for proper cellular signaling. Conversely, mitochondrial ATP ensures the proper functioning of SR Ca2+-handling proteins, which ensures that mitochondria receive an adequate supply of Ca2+. Recent evidence suggests that altered SR Ca2+ proteins, such as ryanodine receptors and the sarco/endoplasmic reticulum Ca2+ ATPase pump, play an important role in maintaining proper cardiac membrane excitability, which may be initiated and potentiated when mitochondria are dysfunctional. This recognized mitochondrial role offers the opportunity to develop new therapeutic approaches aimed at preventing cardiac arrhythmias in cardiac disease.

Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete ß-adrenergic response.

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via ß-adrenergic receptor (ß-AR) activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent "hyperphosphorylation" of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation-contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch-clamp recordings and confocal Ca2+ imaging during ß-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (I Ca) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during ß-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

Atrial Infarction-Induced Spontaneous Focal Discharges and Atrial Fibrillation in Sheep: Role of Dantrolene-Sensitive Aberrant Ryanodine Receptor Calcium Release.

BACKGROUND: The mechanisms underlying spontaneous atrial fibrillation (AF) associated with atrial ischemia/infarction are incompletely elucidated. Here, we investigate the mechanisms underlying spontaneous AF in an ovine model of left atrial myocardial infarction (LAMI). METHODS AND RESULTS: LAMI was created by ligating the atrial branch of the left anterior descending coronary artery. ECG loop recorders were implanted to monitor AF episodes. In 7 sheep, dantrolene-a ryanodine receptor blocker-was administered in vivo during the 8-day observation period (LAMI-D, 2.5 mg/kg, IV, BID). LAMI animals experienced numerous spontaneous AF episodes during the 8-day monitoring period that were suppressed by dantrolene (LAMI, 26.1±5.1; sham, 4.3±1.1; LAMI-D, 2.8±0.8; mean±SEM episodes per sheep, P<0.01). Optical mapping showed spontaneous focal discharges (SFDs) originating from the ischemic/normal-zone border. SFDs were calcium driven, rate dependent, and enhanced by isoproterenol (0.03 µmol/L, from 210±87 to 3816±1450, SFDs per sheep) but suppressed by dantrolene (to 55.8±32.8, SFDs per sheep, mean±SEM). SFDs initiated AF-maintaining reentrant rotors anchored by marked conduction delays at the ischemic/normal-zone border. NOS1 (NO synthase-1) protein expression decreased in ischemic zone myocytes, whereas NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase and xanthine oxidase enzyme activities and reactive oxygen species (DCF [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate]-fluorescence) increased. CaM (calmodulin) aberrantly increased [3H]ryanodine binding to cardiac RyR2 (ryanodine receptors) in the ischemic zone. Dantrolene restored the physiological binding of CaM to RyR2. CONCLUSIONS: Atrial ischemia causes spontaneous AF episodes in sheep, caused by SFDs that initiate reentry. Nitroso-redox imbalance in the ischemic zone is associated with intense reactive oxygen species production and altered RyR2 responses to CaM. Dantrolene administration normalizes the CaM response, prevents LAMI-related SFDs, and AF initiation. These findings provide novel insights into the mechanisms underlying ischemia-related atrial arrhythmias.

Eplerenone Reduces Atrial Fibrillation Burden Without Preventing Atrial Electrical Remodeling.

BACKGROUND: The aldosterone inhibitor eplerenone (EPL) has been shown to reduce the incidence of atrial fibrillation (AF) in patients with systolic heart failure, but the mechanism is unknown. OBJECTIVES: This study hypothesized that by reducing atrial dilation and fibrosis in the absence of heart failure, EPL also reduces AF burden and prevents AF perpetuation. METHODS: The authors conducted a randomized controlled study in 34 sheep that were atrially tachypaced (13 ± 1 week). They compared daily oral EPL (n = 19) versus sugar pill (SP) treatment (n = 15) from the start of tachypacing. The endpoint was a continuous 7-day stretch of persistent AF (n = 29) or completion of 23 weeks tachypacing (n = 5). RESULTS: EPL significantly reduced the rate of left atrial dilation increase during AF progression. Atria from EPL-treated sheep had less smooth muscle actin protein, collagen-III expression, interstitial atrial fibrosis, and cell hypertrophy than SP-treated sheep atria did. However, EPL did not modify the AF-induced increase in the rate of dominant frequency and ion channel densities seen under SP treatment, but rather prolonged the time to persistent AF in 26% of animals. It also reduced the degree of fibrillatory conduction, AF inducibility, and AF burden. CONCLUSIONS: In the sheep model, EPL mitigates fibrosis and atrial dilation, modifies AF inducibility and AF complexity, and prolongs the transition to persistent AF in 26% of animals, but it does not prevent AF-induced electrical remodeling or AF persistence. The results highlight structural remodeling as a central upstream target to reduce AF burden, and the need to prevent electrical remodeling to avert AF perpetuation.

Tbx20 controls the expression of the KCNH2 gene and of hERG channels.

Long QT syndrome (LQTS) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. We functionally analyzed the KCNH2 (encoding for Kv11.1 or hERG channels) and TBX20 (encoding for the transcription factor Tbx20) variants found by next-generation sequencing in two siblings with LQTS in a Spanish family of African ancestry. Affected relatives harbor a heterozygous mutation in KCNH2 that encodes for p.T152HfsX180 Kv11.1 (hERG). This peptide, by itself, failed to generate any current when transfected into Chinese hamster ovary (CHO) cells but, surprisingly, exerted "chaperone-like" effects over native hERG channels in both CHO cells and mouse atrial-derived HL-1 cells. Therefore, heterozygous transfection of native (WT) and p.T152HfsX180 hERG channels generated a current that was indistinguishable from that generated by WT channels alone. Some affected relatives also harbor the p.R311C mutation in Tbx20. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Tbx20 enhanced human KCNH2 gene expression and hERG currents (IhERG) and shortened action-potential duration (APD). However, Tbx20 did not modify the expression or activity of any other channel involved in ventricular repolarization. Conversely, p.R311C Tbx20 did not increase KCNH2 expression in hiPSC-CMs, which led to decreased IhERG and increased APD. Our results suggest that Tbx20 controls the expression of hERG channels responsible for the rapid component of the delayed rectifier current. On the contrary, p.R311C Tbx20 specifically disables the Tbx20 protranscriptional activity over KCNH2 Therefore, TBX20 can be considered a KCNH2-modifying gene.

Scn2b Deletion in Mice Results in Ventricular and Atrial Arrhythmias.

BACKGROUND: Mutations in SCN2B, encoding voltage-gated sodium channel ß2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that ß2-subunits play critical roles in the establishment or maintenance of normal cardiac electric activity in vivo. METHODS AND RESULTS: To understand the pathophysiological roles of ß2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current densities in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional reentry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild-type ventricles, as were levels of Nav1.5 protein, suggesting that similar to the previous work in neurons, the major function of ß2-subunits in the ventricle is to chaperone voltage-gated sodium channel α-subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared with wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than wild-type littermates. CONCLUSIONS: Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.

Low Proteolytic Clipping of Histone H3 in Cervical Cancer.

Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC.

Galectin-3 Regulates Atrial Fibrillation Remodeling and Predicts Catheter Ablation Outcomes.

OBJECTIVES: To determine whether Gal-3 mediates sustained atrial fibrillation (AF)-induced atrial structural and electrical remodeling and contributes to AF perpetuation. BACKGROUND: Galectin-3 (Gal-3) mediates extracellular matrix remodeling in heart failure, but its role in AF progression remains unexplored. METHODS: We examined intracardiac blood samples from patients with AF (N=55) to identify potential biomarkers of AF recurrence. In a sheep model of tachypacing-induced AF (N=20), we tested the effects of Gal-3 inhibition during AF progression. RESULTS: In patients, intracardiac serum Gal-3 levels were greater in persistent than paroxysmal AF and independently predicted atrial tachyarrhythmia recurrences after a single ablation procedure. In the sheep model, both Gal-3 and TGF-ß1 were elevated in the atria of persistent AF animals. The Gal-3 inhibitor GM-CT-01 (GMCT) reduced both Gal-3 and TGF-ß1-induced sheep atrial fibroblast migration and proliferation in vitro. GMCT (12 mg/kg twice/week) prevented the increase in serum procollagen type III N-terminal peptide seen during progression to persistent AF, and also mitigated atrial dilatation, myocyte hypertrophy, fibrosis, and the expected increase in dominant frequency of excitation. Atria of GMCT-treated animals had significantly less TGF-ß1-Smad2/3 signaling pathway activation and expression of α-smooth muscle actin and collagen than saline-treated animals. Ex-vivo hearts from GMCT-treated animals had significantly longer action potential durations and fewer rotors and wavebreaks during AF, and myocytes had lower functional expression of inward rectifier K+ channel (Kir2.3) than saline-treated animals. Importantly, GMCT increased the probability of spontaneous AF termination, decreased AF inducibility and reduced overall AF burden. CONCLUSIONS: Inhibiting Gal-3 during AF progression might be useful as an adjuvant treatment to improve outcomes of catheter ablation for persistent AF. Gal-3 inhibition may be a potential new upstream therapy for prevention of AF progression.

MDIMP, a novel cardiac Ca(2+) channel blocker with atrial selectivity.

In cardiac muscle cells both T-and L-type Ca(2+) channels (TTCCs and LTCCs, respectively) are expressed, and the latter are relevant to a process known as excitation-contraction coupling (ECC). Evidence obtained from docking studies suggests that isoindolines derived from α-amino acids bind to the LTCC CaV1.2. In the present study, we investigated whether methyl (S)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate (MDIMP), which is derived from L-leucine, modulates both Ca(2+) channels and ECC. To this end, mechanical properties, as well as Ca(2+) transients and currents, were all investigated in isolated cardiac myocytes. The effects of MDIMP on CaV1.2 (transiently expressed in 293T/17 cells) were also studied. In this system, evidence was found for an inhibitory action that develops and recovers in min, with an IC50 of 450µM. With respect to myocytes: atrial-TTCCs, atrial-LTCCs, and ventricular-LTCCs were also inhibited, in that order of potency. Accordingly, Ca(2+) transients, contractions, and window currents of LTCCs were all reduced more strongly in atrial cells. Interestingly, while the modulation of LTCCs was state-independent in these cells, it was state-dependent, and dual, on the ventricular ones. Furthermore, practically all of the ventricular LTCCs were closed at resting membrane potentials. This could explain their resistance to MDIMP, as they were affected in only open or inactivated states. All these features in turn explain the preferential down-regulation of the atrial ECC. Thus, our results support the view that isoindolines bind to Ca(2+) channels, improve our knowledge of the corresponding structure-function relationship, and may be relevant for conditions where decreased atrial activity is desired.

Functional and structural impact of pirfenidone on the alterations of cardiac disease and diabetes mellitus.

A synthetic compound, termed pirfenidone (PFD), is considered promising for the treatment of cardiac disease. It leads to beneficial effects in animal models of diabetes mellitus (DM); as well as in heart attack, atrial fibrillation, muscular dystrophy, and diabetic cardiomyopathy (DC). The latter is a result of alterations linked to metabolic syndrome as they promote cardiac hypertrophy, fibrosis and contractile dysfunction. Although reduced level of fibrosis and stiffness represent an essential step in the mechanism of PFD action, a wide range of functional effects might also contribute to the therapeutic benefits. For example, PFD stimulates L-type voltage-gated Ca(2+) channels (LTCCs), which are pivotal for a process known as excitation-contraction coupling (ECC). Recent evidence suggests that these two types of actions - namely structural and functional - aid in treating both cardiac disease and DM. This view is supported by the fact that in DC, for example, systolic dysfunction arises from both cardiac stiffness linked to fibrosis and down-regulation of ECC. Thus, not surprisingly, clinical trials have been conducted with PFD in the settings of DM, for treating not only cardiac but also renal disease. This review presents all these concepts, along with the possible mechanisms and pathophysiological consequences.

Chronic potentiation of cardiac L-type Ca(2+) channels by pirfenidone.

AIMS: On the basis of its ability to inhibit fibrosis, pirfenidone has drawn the attention as an intriguing candidate for treating cardiac disease. However, its precise electrophysiological effects have yet to be elucidated. Here, we have investigated its potential to modulate ion channels. METHODS AND RESULTS: Adult rat cardiac myocytes were investigated using whole-cell patch-clamp, western-blot and qRT-PCR techniques. Pirfenidone increased the density of L-type Ca(2+) current (I(CaL,) 50-100%), without significantly altering Na(+), K(+), or T-type Ca(2+) currents. The effect was dose-dependent, with an EC(50) of 2.8 µM. Its onset was slow, with a lag period larger than 1 h and time to maximum of 24-48 h. Concomitant changes were observed in the voltage-dependent activation of I(CaL) (-5 mV shift in both V(1/2) and k). In contrast, the following properties of I(CaL) remained normal: steady-state inactivation, Ca(V)1.2 levels (mRNA and protein), and intramembrane charge movement. Indeed, the conductance-to-charge ratio, or G(max)/Q(max), was increased by 80%. The effect on I(CaL) was mimicked by an inhibitor of nitric oxide (NO) synthase (NOS), and attenuated by both cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) inhibitors. Conversely, cytokines, reactive oxygen species, and Ca(2+) were all ruled out as possible intermediaries. Additional experiments suggest that pirfenidone increases action potential duration by ∼50%. CONCLUSION: Pirfenidone augments I(CaL), not through higher expression of L-type channels, but through promoting their Ca(2+)-conducting activity. A possible inhibition of NOS expression is likely involved, with subsequent reduced NO production and stimulated cAMP/PKA signalling. These findings may be relevant to the cardioprotective effect of pirfenidone.

Calcitonin gene-related peptide restores disrupted excitation-contraction coupling in myotubes expressing central core disease mutations in RyR1.

Central core disease (CCD) is a congenital human myopathy associated with mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), resulting in skeletal muscle weakness and lower limb deformities. The muscle weakness can be at least partially explained by a reduced magnitude of voltage-gated Ca(2+) release (VGCR). To date, only a few studies have focused on identifying potential therapeutic agents for CCD. Therefore, in this work we investigated the potential use of the calcitonin gene related peptide (CGRP) to restore VGCR in myotubes expressing CCD RyR1 mutants. We also examined the influence of CCD mutants on Ca(2+)-dependent processes involved in myogenesis (myoblast fusion and sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2) gene expression). C2C12 cells were transfected with cDNAs encoding either wild-type RyR1 or CCD mutants, and then exposed to CGRP (100 nm, 1-4 h). Expression of the I4897T mutant significantly inhibited SERCA2 gene expression and myoblast fusion, whereas the Y523S mutant exerted the opposite effect. Interestingly, both mutants clearly inhibited VGCR (50%), due to a reduction in SR Ca(2+) content. However, no major changes due to CGRP or CCD mutants were observed in I(CaL). Our data suggest that the Y523S mutant results in store depletion via decompensated SR Ca(2+) leak, while the I4897T mutant inhibits SERCA2 gene expression. Remarkably, in both cases CGRP restored VGCR, likely to have been by enhancing phospholamban (PLB) phosphorylation, SERCA activity and SR Ca(2+) content. Taken together, our data show that in the C2C12 model system, changes in excitation-contraction coupling induced by the expression of RyR1 channels bearing CCD mutations Y523S or I4897T can be reversed by CGRP.

Long-term modulation of Na+ and K+ channels by TGF-ß1 in neonatal rat cardiac myocytes.

Previous work shows that transforming growth factor-ß1 (TGF-ß1) promotes several heart alterations, including atrial fibrillation (AF). In this work, we hypothesized that these effects might be associated with a potential modulation of Na(+) and K(+) channels. Atrial myocytes were cultured 1-2 days under either control conditions, or the presence of TGF-ß1. Subsequently, Na(+) (I(Na)) and K(+) (I(K)) currents were investigated under whole-cell patch-clamp conditions. Three K(+) currents were isolated: inward rectifier (I(Kin)), outward transitory (I(to)), and outward sustained (I(Ksus)). Interestingly, TGF-ß1 decreased (50%) the densities of I(Kin) and I(Ksus) but not of I(to). In addition, the growth factor reduced by 80% the amount of I(Na) available at -80 mV. This effect was due to a significant reduction (30%) in the maximum I(Na) recruited at very negative potentials or I(max), as well as to an increased fraction of inactivated Na(+) channels. The latter effect was, in turn, associated to a -7 mV shift in V(1/2) of inactivation. TGF-ß1 also reduced by 60% the maximum amount of intramembrane charge movement of Na(+) channels or Q(max), but did not affect the corresponding voltage dependence of activation. This suggests that TGF-ß1 promotes loss of Na(+) channels from the plasma membrane. Moreover, TGF-ß1 also reduced (50%) the expression of the principal subunit of Na(+) channels, as indicated by western blot analysis. Thus, TGF-ß1 inhibits the expression of Na(+) channels, as well as the activity of K(+) channels that give rise to I(Ksus) and I(Kin). These results may contribute to explaining the previously observed proarrhythmic effects of TGF-ß1.

Role of TGF-beta on cardiac structural and electrical remodeling.

The type beta transforming growth factors (TGF-betas) are involved in a number of human diseases, including heart failure and myocardial arrhythmias. In fact, during the last 20 years numerous studies have demonstrated that TGF-beta affects the architecture of the heart under both normal and pathological conditions. Moreover, TGF-beta signaling is currently under investigation, with the aim of discovering potential therapeutic roles in human disease. In contrast, only few studies have investigated whether TGF-beta affects electrophysiological properties of the heart. This fact is surprising since electrical remodeling represents an important substrate for cardiac disease. This review discusses the potential role of TGF-beta on cardiac excitation-contraction (EC) coupling, action potentials, and ion channels. We also discuss the effects of TGF-beta on cardiac development and disease from structural and electrophysiological points of view.

Sustained CGRP1 receptor stimulation modulates development of EC coupling by cAMP/PKA signalling pathway in mouse skeletal myotubes.

We investigated modulation of excitation-contraction (EC) coupling by calcitonin gene-related peptide (CGRP), which is released by motorneurons during neuromuscular transmission. Mouse skeletal myotubes were cultured either under control conditions or in the presence of 100 nm CGRP ( approximately 4-72 h). T- and L-type Ca(2+) currents, immobilization resistant charge movement, and intracellular Ca(2+) transients were characterized in whole-cell patch-clamp experiments. CGRP treatment increased the amplitude of voltage-gated Ca(2+) release ((DeltaF/F)(max)) approximately 75-350% and moderately increased both maximal L-current conductance (G(max)) and charge movement (Q(max)). In contrast, CGRP treatment did not affect their corresponding voltage dependence of activation (V(1/2) and k) or T-current density. CGRP treatment enhanced voltage-gated Ca(2+) release in approximately 4 h, whereas the effect on L-channel magnitude took longer to develop ( approximately 24 h), suggesting that short-term potentiation of EC coupling may lead to subsequent long-term up-regulation of DHPR expression. CGRP treatment also drastically increased caffeine-induced Ca(2+) release in approximately 4 h ( approximately 400%). Thus, short-term potentiation of EC coupling is due to an increase in sarcoplasmic reticulum Ca(2+) content. Both application of a phosphodiesterase inhibitor (papaverine) and a membrane-permeant cAMP analogue (Db-cAMP) produced a similar potentiation of EC coupling. Conversely, this potentiation was prevented by pretreatment with either CGRP1 receptor antagonist (CGRP(8-37)) or a PKA inhibitor (H-89). Thus, CGRP acts through CGRP1 receptors and the cAMP/PKA signalling pathway to enhance voltage-gated Ca(2+) release. Effects of CGRP on both EC coupling and L-channels were attenuated at later times during myotube differentiation. Therefore, we conclude that CGRP accelerates maturation of EC coupling.